According to a known method of tracking a movement of a single molecule in a sample, the molecule is excited with light for emitting photons, and the photons emitted by the molecule are detected with a two-dimensional detector imaging the sample. The current position of the molecule is then determined from the spatial distribution of the photons detected by the detector. With an appropriate pixel density of the detector, a present position of the molecule can be determined from the spatial distribution of the photons at a spatial resolution or precision surpassing the diffraction limit. However, it is a precondition of tracking the particle at a spatial precision beyond the diffraction limit that a high number of photons is detected for each position of the molecule, i.e. before the molecule changes its position. This is due to the fact that a higher number of photons enhances the spatial precision achieved in determining the position of the molecule, only if this position remains unchanged for the whole period over which the higher number of photons are emitted by the molecule.
The spatial precision is given by the radius Δr of a circle around a position of the molecule determined from the centre of intensity of a distribution of positions at which the photons emitted by the molecule are detected by a two-dimensional detector. The true position of the molecule is located within that circle. The radius Δr is given byΔr=FWHM/N1/2   (1)and depends on the number N of detected photons and on the full width at half maximum FWHM of the diffraction pattern.
As the known method of tracking a movement of a single molecule requires a huge number N of photons for each position of the molecule in the sample, the molecule is seriously stressed which results in an increased risk of bleaching the molecule. In the process of bleaching, the molecule chemically changes such that no more photons are provided by the molecule after bleaching. Besides photochemical bleaching, it is also possible that a molecule which has been intensively and/or numerously excited to emit photons is transferred into a metastable dark state. From the metastable dark state the molecule may return after some time. In the metastable dark state, however, the molecule does not emit any photons required for continuously tracking the molecule.
Consequently, there are only some molecules, i.e. only some so-called fluorescent dyes or fluorophores, which are suited for use in the known method. Many fluorophores bleach too fast and hence their movement or the movement of a molecule marked with the fluorophore cannot be tracked for an extended period of time or a longer distance covered within the sample.
In the method described above, the position of the molecule is determined from the distribution of positions at which the photons emitted by the molecule are detected by a two-dimensional detector. This approach is called localization. Another approach for achieving a high resolution or precision in determining a spatial position of photon emitting molecules is so-called STED or RESOLFT fluorescence microscopy. Here, the spatial region in which the molecules in a sample are effectively excited to emit photons is reduced to a size smaller than the diffraction limit. Thus, the photons emitted from the sample can be attributed to this particular spatial region of reduced size independent from the position where the photons are detected and independent from the number of photons detected. In practice, the reduction of the spatial region of effectively exciting the molecules to emit photons is achieved by applying a focused excitation light beam which is superimposed with an interference pattern of one or more coherent beams of fluorescence inhibiting light. This interference pattern comprises a point of essentially zero intensity in the focal region of the excitation light beam. For high absolute intensities of the beams of fluorescence inhibiting light, the intensity of the fluorescence inhibiting light exceeds a saturation intensity IS anywhere but at the point of essentially zero intensity such that the emission of photons by the molecules in the sample is inhibited essentially anywhere but at the point of essentially zero intensity. The achieved spatial resolution or precision is given byΔr=λ(n sin α(1+I/IS)1/2)   (2),wherein I is the maximal intensity of the interference pattern in the sample.
In STED fluorescence microscopy the inhibition of fluorescence is achieved by stimulated emission. In case of RESOLFT fluorescence microscopy the inhibition of fluorescence is achieved by temporarily transferring the molecules into a conformational or other type of state in which the molecules are not capable to fluoresce. Since in STED fluorescence microscopy high absolute intensities of the fluorescence inhibiting light are required, the risk of bleaching the fluorophores is relatively high. For RESOLFT fluorescence microscopy, relatively low intensities of the fluorescence inhibiting light are sufficient. However, this approach can only be applied with special fluorophores that can be switched into a conformational or other type of state in which the fluorophores are not capable to fluoresce.
In general, approaches like a STED or RESOLFT fluorescence microscopy would be suited for tracking a movement of a particle in a sample, in that the particle is tracked with the region of spatially reduced size where the particle is effectively excited to emit fluorescence light. In this case, the criterion for the particle being in the region of spatially reduced size would be a maximum rate of photons emitted by the tracked particle. Although less photons would be required for tracking according to this approach than for continuous localization of the particle, the number of particles and markers which are suited for tracking a movement over longer distances could not be significantly increased. Besides, in STED and RESOLFT fluorescence microscopy, different light beams have to be applied for providing the excitation light and the light for inhibiting fluorescence. Typically, this requires additional effort since the different light beams have different wavelengths and since the different light beams have to be carefully aligned spatially.
From DE 25 46 952 A1 it is known that an optical system based on so-called attenuated total reflectance may be applied to track movements of particles in a sample. According to DE 25 46 952 A1 the sample is subjected to light driving the particles to emit photons. Since the intensity distribution of the light illuminating the sample is not homogeneous but spatially modulated, a movement of the particle results in a respective fluctuation of the number of emitted photons. Thus, considering the modulation of the intensity distribution, the movement of the particle can be concluded from the detected fluctuation of the emitted photons, i.e. the modulation of a detector signal. However, a movement of a particle moving along a path of constant light intensity cannot be tracked. Further, a particle never or only rarely subjected to the light during its movement cannot be tracked at all. Thus, it is essential for tracking the movements of particles in a sample with the optical system known from DE 25 46 952 A1 that the particles are frequently subjected to the light. Hence, the risk of bleaching the particles or markers marking the particles is not effectively diminished but has to be accepted.
There still is a need of a method of and an apparatus for tracking a movement of a particle in a sample in which the risk of bleaching the particle or the marker marking the particle remains small, even if the movement of the particle is tracked for a longer period of time or over a longer distance.